The major histocompatibility (MHC) antigens are polymorphic cell surface glycoproteins with central roles in the generation of immune responses. The class I molecules, which were originally described as the classical transplantation antigens involved in allograft tissue rejection, also present antigens from virally infected, chemically modified, and tumor cells to cytotoxic T cells (CTL). Proper recognition of class I complexes is critical for the initiation of T cell mediated-responses. The goals of the proposed studies are to analyze the structural characteristics of MHC molecules required in the generation of T cell responses as well as to study the influence of soluble analogs of MHC molecules on T cell responses. Preliminary work using cells expressing empty MHC, has shown that certain monoclonal antibody (mAb) serological epitopes associated with the alpha2 domain of the MHC molecule are peptide dependent. Some of the peptide dependent differences are related to changes in the conformational structure of the MHC molecule at the cell surface using fluorescence resonance energy transfer (FRET). To extend these observations the effect of additional peptides on other alpha1 and alpha2 serological epitopes in both empty murine class I MHC molecules and empty human class I HLA molecules will be determined. FRET will be used to analyze the effect of peptide binding on MHC conformational structure as well as to determine the relationship of different domains of the MHC molecule to each other in "empty" MHC molecules versus that seen in peptide loaded MHC molecules. HPLC and microsequencing will be used to define motifs associated with specific mAb precipitated MHC molecules from normal cells line which express peptide loaded MHC molecules. Recent studies have shown that certain regions of the MHC molecule are important in binding antigenic peptides. Site-directed mutagenesis was used to alter three amino acid residues in the C pocket area of the peptide binding region of H-2K/b. These mutated MHC molecules have different peptide binding characteristics. To further characterize the importance of specific amino acid residues, the individual amino acid residue substitutions will be made in H-2K/b and characterized for peptide binding. In addition site-directed mutagenesis will be used to change the peptide-binding region of H-2K/b into the peptide binding region of HLA-A2. The chimeric molecule will be examined for peptide binding and characterized in terms of peptide binding motifs. Preliminary work has shown that soluble divalent MHC can inhibit specific T cell activation. Our focus over the next several years will be to further characterize the soluble divalent MHC molecules. This will include a detailed analysis of: the peptides bound to divalent MHC; the mechanism of inhibition of in vitro allogeneic T cell responses; the influence of specific-peptide loaded divalent MHC on antigen-specific T cell responses; and the influence of divalent MHC on in vivo T cell responses. In addition two additional approaches to making soluble multimeric arrays of MHC proteins which will also be tested as well as their effects on T cell responses. While we do not know if H-2K/b/IgG will inhibit in vivo alloreactive T cell responses, the potency and specificity of inhibition of T cell responses make it a potentially valuable reagent in selectively suppressing undesired MHC-specific T cell responses, as seen in transplantation rejection and autoimmune diseases. To study the effects of H-2K/b/IgG on in vivo T cell activation we will analyze the ability of H-2K/b/IgG to inhibit allograft tissue rejection. An enhanced understanding of the interactions of MHC molecules will improve our understanding of the role of MHC antigens in the generation of both normal and pathological immune responses. Our work with divalent MHC may also provide a potential novel immune based therapeutic reagent.